Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
  • C12M41/14—Incubators; Climatic chambers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
  • C12M37/02—Filters
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/809—Incubators or racks or holders for culture plates or containers

Definitions

• the invention relates to a process and an apparatus for the fermentational production of biologically active materials wherein the fermenter is located in an insulator which in turn is located in a working chamber or is adjacent to it, and wherein a pressure gradient in relation to ambient pressure prevails in both the insulator and in the working chamber.
• the routine construction of facilities for the manufacture of ultrapure compounds is composed of a first, internal chamber which is outwards closed and which is surrounded by a second chamber. In the second chamber the persons working with the biological material are residing, while the processing of the biological material occurs mainly in the first chamber. Both chambers are connected with each other through lockable sluices.
• the first chamber is typically a glovebox so that the process steps may be accomplished from the second chamber with the aid of gloves.
• the second chamber is typically insulated from the outside, but connected to it through lockable sluices.
• both chambers there are ideally sterile conditions which satisfies clean room requirements.
• an over pressure prevails in the first chamber, which is typically an over pressure of 15 Pa.
• the second chamber there exists a low pressure compared to the first chamber, but an over pressure in relation to the ambient pressure. Therefore, particles or germs from the second chamber will be prevented from infiltrating the first chamber. Thus, the biologically active material will be protected against contamination from the environment.
• aerosols from the first chamber may infiltrate the second chamber and can lead to a threat for the persons working in the second chamber. Nevertheless, ensure operational safety, more precautions in this case must be set, as for example vaccination of the employees, wearing of protective clothing, etc.
• an object of the invention is to provide a process and an apparatus for production of biologically active materials by fermentation, wherein the materials will be protected against contamination from the environment and at the same time the exposure of the personnel to aerosols from the fermentation steps and processing of biological material will be reduced or completely prevented.
• the present invention solves this object by providing the apparatus and the process as described herein.
• the invention inter alia comprises the following, alone or in combination:
• An apparatus for the fermentative production of a biologically active compound comprising at least a first insulator containing a fermenter, the first insulator being surrounded or adjacent to a working chamber, wherein the working chamber is connected to the environment via a pressure sluice, wherein a low pressure prevails in the insulator and in the working chamber, wherein the pressure (in relation to ambient pressure) in the insulator is lower than the pressure (in relation to ambient pressure) in the working chamber, and wherein an over pressure prevails in the pressure sluice in relation to ambient pressure, such an
• the apparatus comprises at least a second insulator, and wherein the second insulator does not contain a fermenter, such an
• the apparatus and/or the first insulator and/or the second insulator comprise at least a supply air duct and an exhaust air duct, wherein the supply air duct and the exhaust air duct comprise a filter, selected from a HEPA filter, such an
• first and the second insulator are connected with each other via a passage, wherein the passage allows for material transport from the first insulator to the second insulator and also allows for material transport from the second insulator to the first insulator, such an
• the apparatus comprises a sterilizing facility and/or a disinfection facility, such an
• the first insulator comprises an anaerobic working fermenter, such an
• the first insulator comprises a filtration facility, such an
• the second insulator comprises an extraction facility, such an
• the biologically active compound is a toxin or another protein obtained from fermentation, selected from a botulinum toxin and a botulinum neurotoxin, such a
• botulinum toxin or the mixture of botulinum toxins is/are a botulinum neurotoxin or a composition of botulinum neurotoxins, such a
• the temperature in the first insulator is in a range of approximately 20° to approximately 50° C. and the temperature in the second insulator is in a range of approximately ⁇ 5° to approximately +25° C., such a
• the present invention relates to an apparatus and a process for the production of biologically active agents via fermentation, in an ultrapure form, wherein contamination of the biological material with particles and germs from the environment will be prevented and, at the same time, the potential hazard to the personnel working with the biological material will be reduced.
• the fermentational process includes at least a step of inoculation.
• the fermentational process includes at least one step which is manually conducted, and may be the step of inoculation.
• the process and the apparatus of the present invention allow for the use of approved production techniques and purification techniques which involve, or which could involve, aerosol formation.
• the process and the apparatus of the present invention reduces the potential for persons who are in charge of the production processes and/or the processing processes to be exposed to aerosol formation.
• the apparatus of the present invention and the process of the present invention reduce the risk of exposure of the environment to aerosols and reduces the risk of exposure for production personnel to the biologically active material, bacteria and/or germs.
• the process and the apparatus of the present invention allows for the application of established techniques for efficient production and purification of a biological material, which may produce an aerosol.
• An insulator is an airtight defined system as compared to the environment that stays in an energetic exchange with its environment, but does not allow for an uncontrolled transport of material with its environment.
• Controlled transport of material with its environment for example the respective load or withdrawal of equipment, reagents, starting material, intermediate products, and end products, can be provided by one or more sluices, double trap container, i. e. so called ⁇ -ports in connection with ⁇ -containers or ⁇ -bags. Controlled transport of material is possible before, during or after the fermentation and/or the purification of the fermentation product or the biologically active material.
• the insulator can be operated at over pressure or low pressure regulated through appropriate facilities. It is likewise possible that an atmosphere of inert gas will be generated in the insulator.
• the insulator typically operates on one or more supply air duct(s) and exhaust air duct(s) which are typically equipped with filters. Thus in the insulator, the number of germs may be less than 100 germs/m 3 , less than 10 germs/m 3 , and less than 1 germ/m 3 .
• filters are known to one of skill in the art.
• An example for such filter is a HEPA (high efficiency particulate filter) filter.
• the equipment, apparatuses, or objects situated in the insulator can be moved or operated via a control system from the outside or via glove inlets attached to the insulator which may be actuated from the working chamber.
• the insulator is preferably made completely or partially from a material which is biologically inert and easy to clean and which allows for an in situ decontamination or sterilization, respectively. Therefore a material is preferred which allows for sterilization with formalin, ethylene oxide or vaporous hydrogen peroxide (VHP).
• materials include, for example, glass, stainless steel or polymers, such as PVC.
• the insulator is manufactured so that the interior surface thereof is completely or partially made of glass, stainless steel or other appropriate materials, such as polymers.
• both hard and also soft polymers hard and soft wall insulators
• Gloves are typically made from neoprene, hypalone, vinyl or other suitable materials.
• the insulator disposes of an installation for temperature control and temperature regulation.
• the insulator is situated in, or abuts to, a working chamber. There is a pressure gradient between the insulator and the working chamber. Thereby, a lower pressure prevails in the insulator compared to the one in the working chamber which, in turn, exhibits a lower pressure compared to ambient pressure.
• the ambient pressure accounts for 101,325 Pa, but may be varying depending on geographical position or meteorological conditions.
• the operating pressure in the insulator is a low pressure in the range of 20 to 200 Pa, in the range of 40 to 100 Pa, and in the range of 55 to 75 Pa, respectively compared to ambient pressure (101,325 Pa).
• the operating pressure in the insulator is approximately 10 to 100 Pa, approximately 20 to 80 Pa, approximately around 40 to 60 Pa lower compared to the working chamber.
• the supply of air into and out of the insulator preferably passes through filters, such that a maximum number of 100 germs/m 3 , a maximum of 10 germs/m 3 or a maximum of 1 germ/m 3 may be in the insulator.
• the working chamber of the present invention is an airtight sealed system which is connected with an airtight seal with the insulator for the purpose of an exchange of matter or equipment through one or more lockable openings. Furthermore, it is connected through at least one lockable pressure sluice for the exchange of matter or equipment and/or acquisition in an airtight manner.
• the working chamber surrounds or abuts to the insulator.
• the supply of air into and out of the working chamber preferably passes through filters, such that a maximum number of germs may be 200 germs/m 3 , a maximum number of 100 germs/m 3 , or a maximum number of 10 germs/m 3 may be in the working chamber.
• filters are for example available in trade under the name HEPA filters.
• an operating pressure prevails which is higher than the one in the insulator, but there exists a pressure gradient in relation to the ambient pressure and to the pressure sluice.
• the pressure in the working chamber is 5 to 50 Pa, 10 to 30 Pa, or 12 to 18 Pa lower than compared to ambient pressure.
• a typical value of the pressure difference, within metrological and equipment dependent fluctuations, is 15 Pa compared to ambient pressure, i.e. the pressure in the working chamber is approximately 15 Pa lower than the ambient pressure.
• the working chamber is connected to the environment via at least one lockable pressure sluice.
• the sluice there is an over pressure in relation to ambient pressure of 10 to 100 Pa, preferably 20 to 80 Pa, and most preferably 25 to 35 Pa.
• a pressure difference exists between the pressure sluice and the working chamber of 10 to 200 Pa, preferably 20 to 100 Pa, and most preferably 40 to 60 Pa.
• a typical value of the pressure difference between the pressure sluice and the working chamber, within metrological and equipment dependant fluctuations, is 45 Pa, i.e. the pressure in the working chamber is approximately 45 Pa lower than the ambient pressure.
• a pressure difference exists between the pressure sluice and the insulator of 10 to 300 Pa, preferably 50 to 200 Pa, and most preferably 80 to 100 Pa.
• the apparatus of the present invention comprises at least one other insulator.
• This at least one other insulator can be situated in the same working chamber as the first insulator, or can abut to the same working chamber, it also can be connected airtightly to the first insulator, for example through one or more lockable sluices, double trap container or ports.
• the insulators need not be connected with each other.
• the at least one other insulator may have the same features as the first insulator which is described above.
• a fermentation step will typically be performed.
• the fermentation step may be one in which a biologically active material will be produced.
• This fermentation step may include one or more inoculations.
• the insulator of the present invention contains at least one fermenter.
• one or more steps or measures which are necessary for the purification of the biological material may also be executed. Therefore fermentation and purification may be executed in one and the same insulator or in different insulators, for example, the fermentation carried out in the first insulator and the purification carried out in the second insulator.
• the fermentation step which may involve the synthesis of a biologically active material, occurs in a fermenter.
• a fermenter Depending on the producing strain which produces the biologically active material, one may selective an anaerobic or an aerobic working fermenter.
• the fermenter is constructed and equipped in accordance with the particular fermentation conditions, in accordance with the knowledge of those skilled in the art.
• the fermenter can be a fermenter for the batch fermentation, the semi-batch fermentation or the continuous fermentation.
• the fermentation step may also comprise the precultivation or the initial cultivation to inoculate the main fermenter.
• the different measures and the corresponding procedural equipment which are required for this purpose are known for those skilled in the art and comprise among other things one or more fermenters which allow a first and/or a second expansion of the stock of the producing strain which produces the biologically active material.
• the measures associated therewith also comprise, for example, the establishment of a working cell bank, the first expansion of cell material, the second expansion of cell material and the actual fermentation step.
• the actual fermentation step concerns the growth of cells for the synthesis of the biologically active compound. These steps may be executed at the optimal temperature or temperature gradient respective of the producing organism. Therefore the temperature will be, if applicable, continuously or discontinuously adapted to the production conditions.
• the insulator contains the equipment necessary for it.
• the purification of the biologically active material may also be conducted in a second insulator of the apparatus of the present invention. As only a part of the measures for purification are conducted in the second insulator, the remaining part of the necessary measurements will be conducted in the first insulator or in one or more further insulators.
• Certain measurements which are involved in the production of biologically active material may also be executed outside of an insulator, particularly those steps not associated with aerosol formation or in instances where the biologically active material is available in a form, or in a container, from which one assumes no hazards for the environment or for the persons engaged with the production or operation.
• the fermentation which generates the biologically active material may include fermentation of bacteria, including bacteria of the Clostridium botulinum family. This is also applicable for recombinant or genetically modified Clostridium botulinum strains as well as for other recombinant or genetically modified heterologous expression systems (e. g. E. coli ).
• the biologically active compound which may be produced by using the fermentation process may be a toxin, including a botulinum toxin and a botulinum neurotoxin.
• Botulinum toxin is produced by the bacterium Clostridium botulinum and exists in different serotypes: botulinum toxin type A, B, C, D, E, F, G.
• the botulinum toxin produced by Clostridium botulinum is a complex of the botulinum neurotoxin, i. e. the protein which is responsible for the toxic effect, and complexing proteins which, as such, are non toxic. These complexing proteins are hemagglutinins with different molar masses, as well as at least one non hemagglutinating protein.
• the complexes from botulinum neurotoxin and the (bacterial) complex proteins are in general termed botulinum toxin.
• the complexes of the types A, B, and C are commercially available, type A for example as BOTOX®. These complexes may be further processed in order to yield complexes of different protein composition and purity, including a botulinum neurotoxin without complexing proteins. Therefore within the scope of this invention the term botulinum neurotoxin will be used to describe the protein which is responsible for the toxic effect, i.e. the botulinum toxin that is free of bacterial complexing proteins.
• the botulinum toxin type A has a molecular mass of approximately 150 kDa and is commercially marketed under the trade name Xeomin® by the applicant.
• botulinum toxin Eligible within the scope of this invention are all forms of the “ botulinum toxin”, especially the different serotypes, the different complexes of botulinum neurotoxin and complex proteins, the botulinum neurotoxin itself, as well as modified or corresponding recombinantly produced botulinum toxins, or botulinum neurotoxins including mutations, deletions etc.
• Such mutant forms may be those which are described in WO 2006/027207 A1.
• mixtures of different serotypes in a complexed and/or uncomplexed and/or recombinant form
• mixtures of botulinum toxins of the types A and B e. g. mixtures of botulinum toxins of the types A and B or mixtures of neurotoxins of the types A and B.
• botulinum neurotoxin of the types A and B is described, for example, in the International Patent Publication No. WO 00/74703.
• the characteristics of the (isolated) botulinum neurotoxin, including being substantially non-immunogenic, are advantageous over a botulinum neurotoxin with complexing proteins, hence there is a tremendous need to provide apparatuses and processes for the safe and reliable industrial production of botulinum neurotoxin, which is provided by the present invention.
• the selection and/or the number of insulators may be selected by those skilled in the art depending on the biological compound to be produced. With the purification described herein in more detail, the use of at least two insulators are advantageously used. Thus the insulators may vary with respect to the temperature which predominates therein or in a portion therein. Thus, within the scope of the present invention, the first insulator will be operated at a higher temperature, e. g. at around 200 to 50° C., and the second insulator will be operated at a lower temperature, for example in the range of about ⁇ 50 to +25° C.
• those steps which necessitate an adequate higher temperature, or which can be executed at this temperature including the steps of production, as well as those of purification, are conducted in the first insulator. That includes especially the inoculation, the fermentation and/or the precipitation of the producing strain.
• those steps of the production process which necessitate an adequate lower temperature, especially for the purification of the neurotoxin are executed in a second insulator and are conducted in a beneficial manner at this lower temperature range.
• the second insulator is in principle similarly built as the first insulator, but is provided with facilities and equipment or technical equipment features according to the apparatus.
• the second insulator may be required for the conduction of the purification steps or for the measures thereof.
• Purification steps which may be conducted in the second insulator are selected from precipitation, extraction, centrifugation, dialysis and chromatography.
• the second insulator may include one or more precipitation facilities, extraction facilities, centrifugation facilities, dialysis facilities and/or chromatography facilities.
• a precipitation facility comprises a reaction container in which the solution for performing the precipitation is contained, equipped with a feeding pipe to add the compound(s) required for precipitation.
• the precipitation facility basically comprises measures for taking out the supernatant as well as the precipitate.
• the present invention is the inclusion of two or more insulators which are connected with each other in such a way that the entirety of the apparatus, which comprises both insulators, contains a supply air duct and an exhaust air duct, and that both insulators are connected with one or more intermediate ducts. Therefore, it is also within the scope of the present invention that the first insulator, as well as the second insulator and/or any further insulator, comprise in each case and independent from each other, a supply air duct and an exhaust air duct.
• the first and the second insulator preferably exhibit an internal pressure which is lower compared to the external pressure. At the same time it is within the scope of the present invention that both insulators exhibit an identical internal pressure. It is, however, also within the scope of the present invention that both insulators exhibit different internal pressures. Therefore, the internal pressure of the insulator may be lowered in instances in which the likelihood of aerosol formation is higher due to the process steps which are performed in the respective insulator.
• the availability of a connection between the insulators in the form of a passage, for example in form of a sluice, between both insulators is advantageous.
• the presence of a sluice ensures that the transfer of both the produced and purified material to the different steps of the production and processing can occur without breaking through the barrier built by the insulator.
• the design of the apparatus and of each of the insulator or at least of one insulator of the apparatus of the present invention with a sterilization device serves the same objective. Similar sterilization devices are known to those skilled in the art and comprise for example a generator for the generation of vaporous hydrogen peroxide. Therefore, the use of vaporous hydrogen peroxide is especially beneficial in view of the under pressure conditions.
• the present invention relates to a process for the fermentational production of a biologically active compound wherein the process comprises a fermentation step for the production of the biologically active compound and a purification step of the biologically active compound, in which the fermentational production and/or the purification will be completely or partially executed in one or more insulators.
• the fermentation step and one or more parts of the purification of the biological material occur in a first insulator, and one or more parts of the purification may occur in a second insulator.
• the fermentation step includes at least an inoculation step which is manually conducted. It is also within the scope of the present invention that further insulators will be used.
• the biologically active material may concern a botulinum toxin, especially a botulinum neurotoxin, as described herein.
• the Clostridium strain which may be used in a fermentation is C. botulinum type A which has been stored in a suitable medium at temperatures which guarantee stability.
• the process described by DasGupta B. R. et al. in Toxicon, vol. 22, No. 3, p. 414 to 424, 1984, may be used. Therefore, 0.5% yeast extract and 0.6% autoclaved yeast paste is added to 2% of the N-Z-amine type A medium, and adjusted to a pH of 7.2 by the addition of 4N NaOH and is autoclaved. To this medium, separately autoclaved glucose (20% by weight per volume) is added, for a final concentration of glucose of 0.5% in the medium. Incubation of the producing strain is done at 37° C.
• the supernatant obtained after centrifugation is subject to an ammonium sulfate precipitation either in the first insulator or in the second insulator, whereby other larger proteins will be removed.
• an ammonium sulfate precipitation step is conducted and subsequently the obtained pellet is re-dissolved and is optionally subjected to a dialysis.
• the extract which has been dialysed, and which is obtained from the pellet is centrifuged again and may be subjected to a succession of chromatography steps with the objective to purify the botulinum neurotoxin, especially to purify to homogeneity.
• the container comprising product will be subject to decontamination and accordingly to sterilization.
• the sterilization may be performed via vaporous hydrogen peroxide (VHP).

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